PROJECT ABSTRACT Allogeneic stem cell transplantation (SCT) is a curative therapy for high-risk acute myeloid leukemia (AML); however, relapse remains the leading cause of death post-SCT. AML cures post-SCT depend upon both the intensity of the conditioning therapy and the ability of donor immune cells to recognize and destroy host leukemia cells via the graft versus leukemia effect (GvL). AML has a low mutational burden and therefore a low number of predicted tumor specific neoantigens. Consequently, GvL is most likely mediated by donor T cell recognition of minor histocompatibility antigens (mHA) presented on leukemia (and/or hematopoietic) cells by MHC class I and II. This general concept has not been tested in detail however, because of limitations in predicting, identifying and then validating the mHA responsible for the GVL effect in any given SCT recipient. To more fully address the role of GvL mHA responses in controlling AML, we will investigate GvL responses in primary human samples to test our central hypothesis that AML relapse after SCT results from insufficient donor T cell responses to GvL mHA. To test this hypothesis, we will leverage a human tissue collection protocol in HLA-matched related donor (MRD) SCTs to measure the global magnitude and diversity of donor derived GvL mHA specific T cell responses in SCT. To accomplish this goal, we will determine the in vitro immunogenicity of computationally predicted and biochemically confirmed (by targeted mass spectrometry) GvL mHA using a combination of ELISpot, single T cell microraft array cytotoxicity, and single T cell microraft array proliferation measurements. We will map the TCR? sequences of the GvL mHA specific T cells onto the TCR? repertoires of alloreactive T cells post-SCT. We will also investigate the intrinsic resistance to GvL mHA specific T cell mediated cytotoxicity in AML blasts that relapse following SCT using our microraft arrays, and we will associate resistance to GvL mHA specific T cell cytotoxicity with differences in transcriptome profiles measured by single cell RNA sequencing. The research in this proposal will provide understanding of the role of GvL mHA in control of AML post- SCT. Because SCT is the most effective form of immunotherapy for AML at this time, our investigations into the magnitude and diversity of mHA targets required to achieve effective AML control could lead to rational improvements in the practice of SCT and immunotherapeutic strategies aimed at enhancing GvL mHA targeting without increasing graft versus host disease risk.